In cardiac excitation-contraction coupling, the sarcoplasmic reticulum (SR) plays an essential role in the regulation of the cytosolic free Ca2+ concentration. There are three major functions of the SR: a) Ca2+-uptake from the cytosol into the SR lumen resulting in muscle relaxation; b) Ca2+ storage in the SR lumen; and c) for these functions are: the Ca2+- transport ATPase (SERCA2), the Ca2+ storage protein calsequestrin and the Ca2+ release channel or ryanodine receptor, respectively. phospholamban (PLB) is another SR protein, which plays a crucial role in the regulation of the Ca2+-ATPase activity and myocardial contractility. In this project, we propose further studies on elucidating the regulatory role of PLB in the mammalian heart and defining the stoichiometric coupling ratio between PLB and the Ca2+-pump, which appears to be a key determinant of cardiac contractile parameters. We also propose to elucidate the role of the PLB phosphorylation status, through regulation of its phosphatase activity by inhibitor-1, in the control of contractility under basal and beta-agonist conditions. Furthermore, we propose to extend our studies to the clinical arena and: a) screen patients with heart failure for point mutations in the areas of interaction between PLB and the SR Ca2+-pump, which may modify the nature or degree of interaction of these two proteins, resulting in pathophysiological consequences; and b) assess the levels of PLB and the SR CA2+ pump as well as the degree of PLB phosphorylation in human failing hearts. Our proposed studies will advance our knowledge on the mechanisms underlying regulation of Ca2+ homeostasis by the SR function in the normal and failing heart. They will also provide valuable insights into the crosstalk between the various SR Ca2+ handling proteins and their regulatory effects on cardiac contractility.